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Gap‐scan deletion analysis of Bacillus subtilis RNase P RNA
Author(s) -
Waugh David S.,
Pace Norman R.
Publication year - 1993
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.7.1.7678561
Subject(s) - rnase p , rna , rnase h , rnase mrp , bacillus subtilis , transfer rna , microbiology and biotechnology , biology , ribonuclease , non coding rna , ribonuclease iii , mutant , rna induced transcriptional silencing , chemistry , biochemistry , gene , genetics , rna interference , bacteria
We carried out an exhaustive deletion analysis of Bacillus subtilis ribonuclease P (RNase P) RNA, seeking sequences that are essential for its catalytic activity. A collection of partially deleted RNase P RNA genes was used to construct templates for synthesis, by in vitro transcription, of circularly permuted RNA molecules that lack various wild‐type sequences. The mutant RNAs were assayed for catalytic activity in vitro, using a precursor of B. subtilis tRNA Asp as a substrate. Gap‐scan deletion analysis revealed that most of the RNase P RNA sequence is important for activity; only two substantive deletions did not dramatically inhibit pre‐tRNA processing in vitro. One part of the molecule (nucleotides 225‐270) seemed particularly sensitive to deletion, but considering a collection of mutants with overlapping deletion gaps, it was possible to remove every residue in the RNase P RNA without completely abolishing its catalytic activity. Thus, the catalytic mechanism of RNase P does not depend absolutely on a single, particular nucleotide or local sequence for activity.— Waugh, D. S., and Pace, N. R. Gap‐scan deletion analysis of Bacillus subtilis RNase P RNA. FASEB J. 7: 188‐195; 1993.